CN111676323A

CN111676323A - LAMP primer group and kit for detecting chicken astrovirus, detection method and application

Info

Publication number: CN111676323A
Application number: CN202010625032.6A
Authority: CN
Inventors: 谢芝勋; 张民秀; 张艳芳; 谢志勤; 谢丽基; 刘加波; 邓显文; 罗思思; 范晴; 曾婷婷; 黄娇玲; 王盛
Original assignee: Guangxi Veterinary Research Institute
Current assignee: Wuhan Jinbian Testing Technology Service Co ltd
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2020-09-18
Anticipated expiration: 2040-07-01
Also published as: CN111676323B

Abstract

The application discloses an LAMP primer group for detecting chicken astrovirus, a kit, a detection method and application, wherein the primer group comprises a primer with a concentration ratio of 16: 1: 8, the nucleotide sequences of the outer primer, the inner primer and the loop primer are shown as SEQ ID No.1-6 in the sequence table in sequence. The kit is added with the WarmStart LAMP color-changing premix solution, and the reaction can be terminated after 20 minutes of reaction at 65 ℃. The method can detect the chicken I type and chicken II type astrovirus simultaneously, can detect 10 copies of nucleic acid within 20min, and has the advantages of higher sensitivity, strong specificity, high detection speed and recognizable reaction result by naked eyes.

Description

LAMP primer group and kit for detecting chicken astrovirus, detection method and application

Technical Field

The application relates to the technical field of molecular biology, in particular to an LAMP primer group for detecting chicken astrovirus, a kit, a detection method and application.

Background

The Chicken Astrovirus (CASTV) belongs to the avian astrovirus genus of the astrovirus family, the infection of chickens with CASTV can cause broiler dysgenesis syndrome, diarrhea enteritis of chicks, nephritis and reduction of laying rate of laying hens, and the virus can be infected to Chicken embryos in a vertical transmission mode, thereby causing certain economic loss to the Chicken industry. The chicken flocks in various places of China are commonly infected with CASTV, and the infection rate reaches more than 45 percent. The genome of CASTV is about 7kb in length and comprises three open reading frames (ORF1a, ORF1b and ORF2), two non-coding regions (UTR) at the 5 ' end and 3 ' end, and a Poly (A) tail at the 33 ' end. The proteins encoded by ORF1a and ORF1b are serine protease and RNA-dependent RNA polymerase, respectively, both of which are non-structural proteins, while ORF2 encodes a structural protein, i.e. the viral capsid structural protein. The current diagnostic detection method aiming at CASTV usually adopts 3 methods: one is based on enzyme linked immunosorbent assay (ELISA); the second is a target gene amplification method based on polymerase chain reaction, including RT-PCR method and fluorescent quantitative PCR method; the third is the CAstV isolate obtained by cell or chick embryo isolation. The former two methods both need to depend on special instruments, and have higher cost; the third method takes a long time and cannot quickly and timely judge the detection result of the CAStV.

The loop-mediated isothermal amplification (LAMP) technology is characterized in that a reaction mixture is amplified under the isothermal condition (60-65 ℃), the method has the characteristics of rapidness, convenience and sensitivity, and the method is applied to detection of various diseases. Compared with the traditional detection methods (ELISA and PCR), the method does not need expensive instruments, only needs a water bath kettle capable of adjusting the temperature, and has the characteristics of simple and rapid operation. CN109055613A and CN108929919A respectively disclose a chicken I and chicken II astrovirus loop-mediated isothermal amplification detection primer group, a kit and a detection method thereof, the two methods can only respectively detect the chicken I and chicken II astrovirus, the reaction time required by the detection method is longer, namely 40min, the detection sensitivity is lower, and the minimum detection limit is 135 copy/mu L; in addition, the results of the methods of CN109055613A and CN108929919A required judgment under an ultraviolet (350-370nm) irradiation device, and visual detection could not be realized.

Disclosure of Invention

The invention provides an LAMP primer group for detecting chicken astrovirus, which comprises outer primers CASTV-F3 and CASTV-B3, inner primers CASTV-FIP and CASTV-BIP, and loop primers CASTV-LF and CASTV-LB, wherein the outer primers, the inner primers and the loop primers are nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.6 in a sequence table in sequence, or nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.6 in the sequence table are substituted and/or deleted and/or added by one or more nucleotides and have the same functions as the nucleotide sequences shown in the sequence table. The invention also provides an LAMP kit for detecting the chicken astrovirus, which comprises the primer group.

Further, the kit also comprises a WarmStart LAMP color-changing premix, and the concentration ratio of the inner primer, the outer primer and the loop primer in the kit is 16: 1: 8.

further, the reagentEach 20ul reaction system of the cassette comprises 2 uL cDNA template, 10 uL 2 × WarmStart LAMP color-changing premix, 32pmol of mixture of inner primer CAStV-FIP and CAStV-BIP, 2pmol of mixture of outer primer CAStV-F3 and CastV-B3, 16pmol of mixture of loop primer CAStV-LF and CAStV-LB, and the balance ddH₂O。

The invention also provides a method for detecting the chicken astrovirus, which adopts the primer group or the kit to carry out the loop-mediated isothermal amplification reaction and is not used for the diagnosis and treatment of diseases.

Further, the reaction process of the loop-mediated isothermal amplification reaction comprises the steps of reacting at 65 ℃ for 20 minutes and inactivating at 80 ℃ for 5 minutes.

The invention also provides application of the primer group, the kit and the method for detecting the chicken astrovirus in simultaneously detecting the chicken I-type astrovirus and the chicken II-type astrovirus.

The beneficial effects of the invention include: in the invention, when the primers are designed, a universal chicken astrovirus isothermal amplification primer set is designed in a conserved region of ORF1a of chicken I-type and chicken II-type astrovirus, and the chicken I-type and chicken II-type astrovirus can be detected simultaneously; the invention designs a pair of loop primers besides the inner and outer primers for isothermal amplification of the chicken astrovirus, the addition of the loop primers accelerates the reaction speed of the isothermal amplification, 10 copies of nucleic acid can be detected within 20min at the optimal reaction temperature and primer concentration, the sensitivity is higher, the specificity is strong, and the detection speed is faster; the invention adopts commercialization

The LAMP color-changing premix can determine the result through naked eyes according to the color change of a reaction product after the reaction is finished, and the detection method is more convenient and faster without ultraviolet irradiation: after the isothermal amplification reaction is finished, the color of a reaction product is changed from original pink to yellow, which indicates that the result is positive, and if the reaction product does not have color change and presents original pink, the result is determined to be negative. The primer group, the kit and the detection method are more convenient to apply in clinical detection.

Drawings

FIG. 1 is a graph showing the reaction temperatures of 62,63,64 and 65 ℃ respectively for a byproduct magnesium phosphate monitored by a real-time turbidimeter at 650nm under different LAMP reaction temperatures, wherein a sample 1 is a chicken astrovirus, and a sample 2 is a negative control;

FIG. 2 is a diagram showing the result of LAMP specificity test, in which sample 1 is NDV cDNA,2 is MDV DNA,3 is REV cDNA,4 is H7 cDNA,5 is H9 cDNA,6 is H5 cDNA,7 is ALV-J cDNA,8 is E coli DNA,9 is CIAV DNA,10 is FAdV-4DNA,11 is AEV cDNA,12 is IBV cDNA,13 is IBDV cDNA,14 is ARV cDNA,15 is positive CAStV control, and 16 is negative control;

FIG. 3 is a result chart of LAMP sensitivity test, in which FIG. 3A is a graph of a magnesium phosphate byproduct monitored by a real-time turbidimeter at 650nm, FIG. 3B is a result chart of color change of a reaction product after the reaction is finished, samples 1-6 are 100 copies/. mu.L template standard, and

samples

7 and 8 are negative controls;

FIG. 4 is a diagram showing the results of LAMP clinical tests, in which samples 1 to 14 are clinical samples, sample 15 is a positive CAStV control, and sample 16 is a negative control.

Detailed Description

The present invention will be further illustrated and described with reference to the following examples, but the examples described are only a part of the examples of the present invention, and not all of the examples. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Wherein the content of the first and second substances,

LAMP color-changing premix was purchased from New England Biolabs (NEB); the Loopamp LA-320C real-time turbidimeter was purchased from Rongyan Biotechnology (China) Co., Ltd; EasyPure Viral DNA/RNA Kit is a DNA/RNA co-extraction Kit purchased from Beijing QuanzijinshengTechnical Co., Ltd; the reverse transcription kit and pMD-18T were purchased from Baori doctor materials technology (Beijing) Co., Ltd; NanoDrop2000 nucleic acid analyzer was purchased from Thermo Fisher scientific; multicolor fluorescence imaging systems are available from BIO-RAD, USA.

Avian Influenza Virus (AIV) H5(Duck/HK/313/78) and the H7 subtype (Duck/HK/47/76) were gifted by hong Kong university; the Infectious Bursal Disease Virus (IBDV) AV6 strain was purchased from the Chinese veterinary drug laboratory; avian myeloencephalitis virus (AEV) AE1163 was gifted by state university of canaldinger, usa; newcastle Disease Virus (NDV) strain GX6/02, subtype H9 AIV (Duck/Guangxi/1/00), Avian Reovirus (ARV) S1133, avian Reticuloendotheliosis virus (REV) strain 2012001-1 (accession No. KC453976), avian Infectious Bronchial Virus (IBV) strain GXIB/02, Marek' S disease virus (MDV) strain 2012006-1 (accession No. KC453976), avian Infectious bronchial virus 4 (3970) strain GXIB 4, FAdV-4 strain FAdV-4-GX2018001, ALV-J2012004-5 (accession No. KC 45821), avian Infectious adenovirus subtype 4 (KC ad nov virus) strain 0604, victory virus strain # gcv-4 (accession No. gcv) strain reserved by veterinarian; the clinical samples used in the experiment are from throat swabs and cloaca swabs collected in a certain scale chicken farm in Guangxi; the CAStV positive sample has been subjected to common PCR and sequencing identification in the laboratory, and the CAStV positive cDNA used in the test is derived from clinical samples.

The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Example 1 design of primer set

According to the conserved sequence of the ORF1a gene of CAStV in GenBank, MEGA 4.0 and Primer Explorer V4 online Primer design software are used for designing LAMP primers, and the external primers are CAStV-F3 and CAStV-B3; the inner primers are CAStV-FIP and CAStV-BIP, the inner primers are F1c + F2, and the inner primers are B1c + B2; the loop primers are CAStV-LF and CAStV-LB. The primer sequences are shown in Table 1, and the inner primer and the outer primer sequences are synthesized by the Daphne Huada Biotech (Shenzhen) Limited.

TABLE 1 primer sequences

Of the three open reading frames of CASTV, ORF1a and ORF1b are relatively conserved, with the ORF1b gene being most conserved and the ORF2 gene being the most variable. At present, PCR and fluorescent quantitative PCR methods for detecting the chicken astrovirus and CN109055613A and CN108929919A all use ORF1b as target genes for amplification. In the study, 7 sets of LAMP primers are designed from conserved regions based on ORF1a, ORF1b and ORF2 genes, only one set of LAMP primers capable of specifically amplifying positive CAStV cDNA (namely the LAMP primer set aiming at ORF1a gene listed in Table 1) is obtained by screening, and the other 6 sets of primers are not amplified.

Example 2 verification of LAMP detection method of Chicken Star Virus

Extraction of RNA/DNA template

RNA and DNA from different viruses and clinical samples were extracted according to the instructions in the EasyPure Viral DNA/RNA Kit, RNA virus was reverse transcribed into cDNA, and the cDNA/DNA template was stored at-20 ℃ for use.

2. Preparation of Positive plasmid Standard

2.1 preparation of Chicken type II Star Virus Standard

And respectively amplifying a truncated target gene by using external primers CASTV-F3 and CASTV-B3 and using a CASTV positive cDNA as a template, wherein the sizes of target fragments are respectively 226bp, and the amplified PCR positive product is connected to a pMD-18T vector to construct a recombinant plasmid, extract a positive plasmid, send the recombinant plasmid to a company for sequencing, and identify the recombinant plasmid as a chicken II type astrovirus through sequencing, wherein the chicken II type astrovirus is marked as CASTV-II. The concentration of the positive recombinant plasmid of CAStV was measured by a NanoDropND-2000 ultraviolet spectrophotometer, and the copy number was calculated from the molecular weight and concentration.

2.2 preparation of Standard Chicken type I Star Virus

Referring to the ORF1a gene sequence of chicken I-type astrovirus sequence CAStV/Poland/G059/2014 (accession number: KT886453) published on GenBank, the ORF1a gene is truncated in the region of 467 th base 242, the truncated gene is sent to a company for gene synthesis, the truncated gene is used as a template for PCR amplification by using the outer primers CAStV-F3 and CAStV-B3, and the chicken I-type astrovirus standard, which is marked as CAStV-I, is prepared according to the steps prepared in the standard 2.1. The concentration of the positive recombinant plasmid of CAStV is measured by a NanoDrop ND-2000 ultraviolet spectrophotometer, and the copy number is calculated according to the molecular weight and the concentration.

Optimization of LAMP reaction System and reaction conditions

mu.L of cDNA template, WarmStart Colorimetric LAMP 2 × Master Mix 10. mu.L, 2. mu.L of mixture of inner primers CASTV-FIP and CASTV-BIP (working concentration of 16. mu. mol/L), 2. mu.L of mixture of outer primers CASTV-F3 and CastV-B3 (working concentration of 2. mu. mol/L), 2. mu.L of mixture of loop primers CASTV-LF and CASTV-LB (working concentration of 8. mu. mol/L), and ddH₂O make up to 20. mu.L. At the same time, a negative control was set up and no RNAase water was added as a template to the reaction tube. Placing the reaction tube in a Loopamp LA-320C real-time turbidimeter, adjusting reaction temperature (62 deg.C, 63 deg.C, 64 deg.C and 65 deg.C), reacting for 60min to determine the optimal reaction temperature, and inactivating at 80 deg.C for 5 min.

The result of the reaction product is judged according to

And (4) judging by the LAMP color-changing premix kit instruction, wherein the color of the LAMP reaction product is changed from pink reaction solution to yellow, the result is positive, and if the color is not changed, the result is negative. At present, the judgment result of the LAMP detection method established by most scholars is mainly judged by two ways of observing the trapezoidal strip by direct gel electrophoresis of a reaction product and adding a fluorescent color developing agent into an amplification product for color development, and the two ways of carrying out subsequent operation after opening a reaction tube cover, so that the reaction product is easy to form aerosol, the experimental environment is polluted, the result is easy to cause false positive, and the judgment on the result is very unfavorable. Although CN109055613A and CN108929919A added fluorescent reagent before reaction avoids pollution caused by uncovering, the reaction result still needs an ultraviolet lampCan be observed only in the next place. Electrophoresis and fluorescent agent addition are not needed after amplification is finished, pollution of reaction products to a laboratory is reduced, and the experimental result can be judged through the color reaction of the products, so that the detection time is greatly shortened.

As shown in fig. 1. When the temperature is 65 ℃, the negative control of the nephelometer plot has no peak and enters the exponential growth phase at the earliest, so the optimal temperature for the reaction is 65 ℃.

After the optimal reaction temperature is determined, optimizing the concentration of the primers according to the final concentrations of the inner primer (CASTV-FIP/CASTV-BIP), the outer primer (CASTV-F3/CASTV-B3) and the loop primer (CASTV-LF/CASTV-LB) in different proportions, wherein the final concentrations (X [ mu ] mol/L) of the primers in different proportions are respectively 1.6:0.1:0.8, 1.6:0.15:0.8, 1.6:0.2:0.8, 2:0.1:0.8, 2:0.15:0.8, 2.4:0.2:0.8 and 2.4:0.25: 0.8; after the reaction is carried out at the optimum reaction temperature, the optimum concentration and ratio of the primers are determined.

The optimal final concentrations of the inner, outer and loop primers were 1.6. mu. mol/L, 0.1. mu. mol/L and 0.8. mu. mol/L, respectively.

According to the reaction system of CastV-LAMP, the CAStV-II and CAStV-I recombinant plasmids prepared in the step 2 are respectively diluted to 10 in a gradient way according to the calculated copy number⁴、10³、10²And 10¹Copy/. mu.L, 2. mu.L of plasmid of each dilution was taken as a template and added to the reaction tube, while a negative control was set up and RNAase-free water was added as a template to the reaction tube. After the reaction tubes are respectively reacted for 20min, 30min, 40min and 60min, inactivation is carried out for 5min at 80 ℃.

Plasmid templates with different dilutions of CASTV-II and CASTV-I in the CastV-LAMP reaction system all obtain positive reactions within 20min of reaction time, namely the concentration of the contained plasmid is 10⁴、10³、10²And 10¹The reaction liquid of the CastV-LAMP reaction system copied/mu L is pink, which shows that the CastV-LAMP reaction system can complete LAMP amplification within 20min at the fastest speed.

4. Specificity verification test

And (3) using the optimal reaction conditions and the optimal reaction system determined in the step (3), adding cDNA or DNA of CAStV, AIV, IBDV, AEV, NDV, ARV, REV, IBV, MDV, FAdV-4, ALV-J, E coli and CIAV as a template into the reaction system, and simultaneously setting up water without RNAase as a negative control to verify the specificity of the primers.

As shown in FIG. 2, the results showed that only the reaction product to which the positive CAStV cDNA was added had a color change (from pink to yellow).

5. Sensitivity verification test

The CAStV-II and CAStV-I recombinant plasmids prepared in the step 2 are respectively diluted to 10 in a gradient way according to the calculated copy number⁵、10⁴、10³、10²、10¹And 10⁰Copies/. mu.L. And (3) according to the optimal reaction condition and the optimal reaction system determined in the step (3), sequentially adding 2 mu L of recombinant plasmid with gradient dilution into a reaction tube, simultaneously setting a negative control, adding RNAase-free water into the reaction tube by taking RNAase-free water as a template, uniformly mixing, placing the mixture into a Loopamp LA-320C real-time turbidimeter for reaction, and observing the color of a reaction product by naked eyes after the test is finished.

As shown in FIG. 3A, the turbidity results monitored by the real-time turbidimeter showed that the minimum detection limits of CASTV-II and CASTV-I were 10¹Copies/. mu.L. Meanwhile, the result of visually observing the color change of the reaction product after the reaction of the real-time turbidimeter is shown in fig. 3B, wherein samples 1 to 5 are yellow in color and samples 6 to 8 are pink in color in fig. 3B, and thus it can be seen that the minimum detection limit of the CAStV is also 10¹Copies/. mu.L, which corresponds to the results of a real-time turbidimeter.

6. Detection test of clinical samples

Using the optimal reaction conditions and the optimal reaction system determined in

step

3, 14 clinical samples were tested. Meanwhile, the PCR method is adopted to verify the detection result, and the specific sequences of the single PCR primers CAStV-F and CAStV-R for detecting CAStV are shown in SEQ ID NO. 7-8. The amplified fragment size is 361 bp; the primers are synthesized by Shenzhen limited of Huada gene biotechnology, and the primer pairs are used for detecting CASTV, and the obtained result is compared with the result of LAMP detection of CASTV established in the research to verify the reliability of the method.

As shown in FIG. 4, the results show that two CAStV samples are positive, namely sample No. 10 (weak positive) and sample No. 13, and the rest samples are negative, and the detection results are compared with the common PCR method for detecting CAStV in the laboratory, and the LAMP detection result of CAStV according to the invention is consistent with the common PCR detection result of CAStV.

Sequence listing

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Claims

1. The LAMP primer group for detecting the chicken astrovirus is characterized by comprising outer primers CASTV-F3 and CASTV-B3, inner primers CASTV-FIP and CASTV-BIP and loop primers CASTV-LF and CASTV-LB, wherein the outer primers, the inner primers and the loop primers are nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.6 in a sequence table in sequence, or the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.6 in the sequence table are subjected to substitution and/or deletion and/or addition of one or more nucleotides, and have the same functions as the nucleotide sequences shown in the sequence table.

2. An LAMP kit for detecting chicken astrovirus, which is characterized by comprising the primer set of claim 1.

3. The LAMP kit for detecting the chicken astrovirus according to claim 2, wherein the kit further comprises a WarmStart LAMP color-changing premix, and/or the concentration ratio of the inner primer, the outer primer and the loop primer in the kit is 16: 1: 8.

4. the LAMP kit for detecting the chicken astrovirus according to claim 2 or 3, wherein each 20ul reaction system of the kit comprises 2 μ L of cDNA template, 10 μ L of 2 × WarmStart LAMP color-changing premix, 32pmol of mixture of inner primers CASTV-FIP and CASTV-BIP, 2pmol of mixture of outer primers CASTV-F3 and CastV-B3, 16pmol of mixture of loop primers CASTV-LF and CASTV-LB, and the balance ddH₂O。

5. A method for detecting chicken astrovirus, which is characterized in that a loop-mediated isothermal amplification reaction is performed by using the primer set or the kit according to any one of claims 1 to 4, and the method is not used for diagnosis and treatment of diseases.

6. The method for detecting chicken astrovirus according to claim 5, wherein the reaction process of the loop-mediated isothermal amplification reaction comprises a reaction at 65 ℃ for 20 minutes.

7. The method for detecting chicken astrovirus according to claim 6, wherein the reaction process of the loop-mediated isothermal amplification reaction further comprises inactivation at 80 ℃ for 5 min.

8. The primer set of claim 1, the kit of any one of claims 2 to 4, and the method for detecting chicken astrovirus of any one of claims 5 to 7, wherein the primer set is used for simultaneously detecting chicken astrovirus type I and chicken astrovirus type II.